BAP1 missense mutation c.2054 A>T (p.E685V) completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V) variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing

Comparative sequence analysis reveals the <i>BAP1 c</i>.<i>2054A</i> (p.E685) is highly conserved.

<p>A. Multi-species comparative genomic analysis of <i>BAP1 c</i>.<i>2054A</i> in ten distantly related species: human, chimpanzee, mouse, rat, dog, cat, rabbit, chicken, xenopus and zebrafish. B. Multiple sequence alignment indicates BAP1 p.E685 is well conserved across the same ten distantly related species.</p

Detection of aberrant splicing products in HMeso01A harboring the <i>BAP1 c</i>.<i>2054 A>T</i> (p.E685V) mutation.

<p>PCR fragments generated from amplification of <i>BAP1</i> exons 14–17 on cDNAs from HMeso01A and 4 controls were separated by agarose gel electrophoresis. Marker: DNA marker PhiX 174-HaeIII digest ladder.</p

Summary of splicing variants cloned from RT-PCR products of HMeso01A harboring the c.2054 A>T (p.Glu685Val) mutation.

<p>A. Retention of introns 14, 15, 16; B. Retention of intron 15, 16; C. Retention of intron 14 and 4 bp deletion (GAAG) at the end of exon 16; D. Retention of intron 16; E. Partial retention of 3’ 69 bp in intron 14 and 4 bp deletion (GAAG) at the end of exon 16; F. Partial retention of 3’ 18 bp in intron 15 and 4 bp deletion (GAAG) at the end of exon 16; G. 4 bp deletion (GAAG) at then end of exon 16 (Δ4). //: partial exon.</p

CDH1 Missense Variant c.1679C>G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5' Splice Site.

Disease-causing germline mutations in CDH1 cause Hereditary Diffuse Gastric Cancer (HDGC). For patients who meet the HDGC screening criteria, the identification and classification of the sequence variants found in CDH1 are critical for risk management of patients. In this report, we describe a germline CDH1 c.1679C>G (p.T560R) variant identified in a 50 year old man who was diagnosed with gastric cancer with a strong family history of gastric cancer (one living brother was diagnosed with gastric cancer at 63 and another brother died of gastric cancer at 45). cDNA analysis, involving fragment analysis and cloning, indicated that the p.T560R mutation created a novel 5' splice donor site, which led to a novel transcript with a 32 nucleotide deletion in exon 11. This abnormal transcript putatively produces a truncated CDH1 protein (E-cadherin) of 575 amino acids instead of 882. We also demonstrated that the variant completely abolishes normal splicing as the mutant allele does not generate any normal transcript. Furthermore, the CDH1 c.1679C>G (p.T560R) variant segregated with gastric cancer in all three family members affected with gastric cancer in this family. These results support the conclusion that CDH1 c.1679C>G (p.T560R) variant is a pathogenic mutation and contributes to HDGC through disruption of normal splicing

Patient pedigree, <i>CDH1</i> mutation and H&E image of diffuse gastric cancer.

<p>(A) The patient (indicated with the arrow head) is a 50 year old man who was diagnosed with gastric cancer at the age of 50. In his generation, three members were diagnosed with gastric cancers (one brother died of gastric cancer at 45, another brother was diagnosed with gastric cancer at 63). All three brothers affected with gastric cancer were determined to have the <i>CDH1 c</i>.<i>1679 C>G</i> p.T560R variant. (B) our patient’s germline <i>CDH1</i> sequencing demonstrating the <i>CDH1 c</i>.<i>1679C>G</i> variant. (C) Haematoxylin and eosin stain (H&E stain) of patient’s biopsy specimen showing infiltrating adenocarcinoma poorly differentiated with mucinous and signet ring cell features. The signet ring cells are characteristic of hereditary diffuse gastric cancer (HDGC). They contain large amount of mucin which pushes the nucleus to the cell periphery (see arrows).</p

<i>CDH1 c</i>.<i>1679 C>G</i> (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.

<p>(A) Sequence adjacent to wild type allele, <i>CDH1 c</i>.<i>1679 C</i>, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, <i>CDH1 c</i>.<i>1679 G</i>, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.</p

<i>In silico</i> prediction of the <i>CDH1</i> p.T560R variant.

<p>Prediction of the potential effects of the <i>CDH1 c</i>.<i>1679 C>G</i> (p.T560R) alteration on normal mRNA splicing. Top panel: reference sequence; bottom panel: mutated sequence. The text at the left of each panel represents the names of the tools used to predict splice site strength. The number ranges indicate the strength of each target sequence in each prediction tool with the upper range being the perfect match. The reference sequence and mutated sequences are shown at the bottom of each panel with nucleotide positions indicated above. Blue vertical bars indicate 5’ (donor) site. The height of each bar is proportional to the maximum possible score computed by the corresponding algorithm.</p